Process for preparing double-coated lactic acid bacteria powder using protein and polysaccharide and product by the same

ABSTRACT

The present invention relates to a process for production of a double-coated lactic acid bacteria culture powders using protein and polysaccharide. The present invention can provide double-coated lactic acid bacteria through homogenization in the state of an aqueous solution and lyophilization process thereby requiring no additional equipment and apparatus.  
     The double-coated lactic acid bacteria powders according to the present invention show improved heat-resistance, acid-resistance, bile-resistance and storage stability, as well as excellent survival rate until reaching the intestines

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a process for preparingdouble-coated lactic acid bacteria powders using protein andpolysaccharide and the product by the same.

[0003] 2. Description of the Related Art

[0004] It is well known that lactic acid bacteria has many physiologicaleffects in improving and activating the functions of the intestinesthereby maintaining good health.

[0005] A process of non-coated lactic acid bacteria powders comprisesthe steps of cultivating the lactic acid bacteria, concentrating theculture and lyophilizing thereof.

[0006] The obtained non-coated powders, however, are weak to air,moisture and/or temperature. Therefore, when the lactic acid bacteriapasses through the stomach after ingestion, almost all the lactic acidbacteria is killed by the gastric juice before reaching the intestine.Also, the non-coated culture powder has difficulty in maintainingstability during storage, distribution and processing for secondaryproducts.

[0007] In order to overcome such problems, proposed have been a methodfor coating the lactic acid bacteria and a micro-encapsulation processusing gelatins, sugars, gums, etc.

[0008] The conventional process for coating the lactic acid bacteriausually comprises the step of introducing a specified coating materialto the dried lactic acid bacteria culture as shown in FIG. 1.

[0009] The lactic acid bacteria is cultivated M1,M2 within an anaerobicfermentation apparatus (herein after a “fermenter”) using a fermentationmedium containing peptones, meat extracts, yeast extracts, glucose andinorganic ions. Such components contained in the medium arewater-soluble and used only for proliferation of the lactic acidbacteria.

[0010] The concentrated cultures M3 are obtained by means of centrifugalseparation or ultra-filtration, the purpose of which is solelyseparation and concentration of the cultures.

[0011] During deep freezing and lyophilization processes M4, acryoprotectant is generally added to prevent the lactic acid bacteriafrom being killed. In turn, a coating composition in an aqueous solutionM5 is applied to the obtained lactic acid bacteria culture, followed bylyophilization.

[0012] Alternatively, a micro-spherical bead type coating compositionmay be applied to the dried lactic acid bacteria culture, followed by anozzle-injection in a micro-encapsulation process.

[0013] Such conventional processes for coating the lactic acid bacteriainclude the steps for introduction of the coating composition ormicro-encapsulation after concentrating and drying the cultures, whichrequires an expensive coating agent and additional steps therebyincreasing production costs.

[0014] Furthermore, materials and individual steps may overlap since thelyophilization process after coating it in a liquid state requires acryoprotectant and a stabilizer to ensure improved viability andstability.

SUMMARY OF THE INVENTION

[0015] It is an object of the present invention to solve the problemsdescribed above and, in particular, to provide a process for preparing adouble-coated lactic acid bacteria culture powders using protein andpolysaccharide.

[0016] It is another object of the present invention to provide a lacticacid bacteria culture powder having enhanced moisture resistance,heat-resistance, acid-resistance and bile acid-resistance therebyimproving stability by a cost-effective and simple process.

[0017] In accordance with the present invention, there is provided aprocess for preparing a double-coated lactic acid bacteria powdercomprising the steps of: preparing a protein aqueous solution; adding aproteolytic enzyme solution to the protein aqueous solution forproteolysis; cultivating a lactic acid bacteria using the proteinaqueous solution; concentrating the lactic acid bacteria culture using acentrifuge wherein the lactic acid bacteria culture is coated with theprotein; introducing the lactic acid bacteria culture into apolysaccharide aqueous solution and mixing it to form a homogenizedsolution; and lyophilizing the homogenized solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a flow chart showing a conventional process forpreparing a coated lactic acid bacteria culture;

[0019]FIG. 2 is a flow chart showing a preferred embodiment according tothe present invention.

[0020]FIG. 3 is a flow chart showing another preferred embodimentaccording to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0021] Preferred embodiments of the process for preparing double-coatedlactic acid bacteria culture powders according to the present inventionusing the protein and the polysaccharide will now be described belowwith reference to FIGS. 2 and 3.

[0022]FIG. 2 provides a flow chart showing an embodiment of the processfor preparing the double-coated lactic acid bacteria powder, wherein aconcentrated bacteria culture is double-coated with protein andpolysaccharides and followed by a lyophilization process of theresultant double-coated bacteria culture to be in freeze dried powderform.

[0023]FIG. 3 illustrates a flow chart for another embodiment of theprocess for preparing the double-coated lactic acid bacteria powderincluding the steps of primarily coating lactic acid bacteria culturewith protein followed by lyophilization, secondly coating the driedcultures with a polysaccharide solution, and finally lyophilizing theresultant double-coated culture.

[0024] According to the present invention, the process for producing thedouble-coated lactic acid bacteria culture powder comprises severalsteps including the proteolysis process of the protein solution,fermentation process of the lactic acid bacteria, the primary-coatingprocess with a protein using a centrifuge and the secondary-coatingprocess with a polysaccharide.

[0025] In the proteolysis step S1, 1 to 10% by weight of protein aqueoussolution including skim milk and/or isolated soybean protein isprepared. A proteolytic enzyme solution is added, in amount of 0.01-1%by weight based on the total weight of the protein, to the proteinaqueous solution for proteolysis.

[0026] The hydrolyzed products obtained after the proteolysis step canbe classified as water-soluble or low molecular weight peptidecomponents for cultivation of lactic acid bacteria and water-semisolubleor high molecular weight peptide components for the coating of a lacticacid bacteria culture.

[0027] For the fermentation(cultivation) step S2, there are added anddissolved 1-5% by weight of glucose, 0.1-1.5% by weight of yeastextract, 0.1-1.5% by weight of concentrated meat extract, 0.01-0.1% byweight of ionic components, such as ammonium citrate, sodium acetate,dipotassium phosphate, magnesium sulfate, manganese sulfate, sodiumchloride and mixtures thereof, into the hydrolyzed protein solution.Then, the lactic acid bacteria is cultivated, after steam-sterilization,in the solution containing protein and other mixtures within ananaerobic fermenter.

[0028] In the protein-coatings step S3, the lactic acid bacteria cultureis separated and concentrated by using a high speed centrifuge at 15,000RPM or more. A coating mechanism occurs during the centrifuge process,wherein residual protein components in the fermented solution aredeposited with the bacteria culture, capturing and enclosing them.

[0029] The hydrolyzed product composition allows proliferation of thelactic acid bacteria, and conditions can be optimized by increasing orreducing the concentration of protein and enzyme considering thephysiological properties of the used lactic acid bacteria. Thehydrolyzed product can also function as cryoprotectants because itencloses the cultures thereby protecting the bacteria cultures fromforming ice-crystals in a deep freezing process.

[0030] For the polysaccharide-coating step S4, prepared are a 1-10% byweight of polysaccharide aqueous solution including 1-10% by weight ofxantan gum, cellulose, levan and/or mixtures thereof, and a 35-45% byweight of cryoprotectant aqueous solution including 1-10% by weight oftrehalose, malto-dextrin, mannitol and a skim milk, the weight of whichis based on the total weight of the concentrated protein-coated bacteriacultures.

[0031] After sterilization under pressure, both of the above solutionsare mixed together with the protein-coated bacteria cultures byagitating the mixtures in a stirrer to form a homogenized solution andthen, freeze-dried.

[0032] Alternatively, a freeze dried bacteria culture powder, which iscoated with protein and lyophilized (S3-1), may be mixed into themixture of the above polysaccharide and cryoprotectant aqueous solutionhomogeneously using a stirrer and then, freeze-dried whereby thebacteria culture is secondly coated with polysaccharide.

[0033] During the lyophilization process, the lactic acid bacteriacultures homogenized in the polysaccharide solution adhere to each otherhaving an excessively dense structure due to the strong adhesiveness ofthe polysaccharide among cultures. The lactic acid bacteria culturepowders have a constant particle size ranging from 40-120 throughgrinding process.

[0034] With regard to properties of the polysaccharide used as thesecondary coating agent in the present invention, the polysaccharide hasan inherent adhesiveness and its hydrophilic properties allow it to becompatible with the primary coating agent, that is, the protein, as wellas other water-soluble sugars, cryoprotectants including amino acid andstabilizers for lactic acid bacteria. In particular, the polysaccharidein an aqueous solution has extremely low solubility under acidicconditions while it can be easily dissociated at neutral or higher pHvalues, whereby the polysaccharide may improve stability and viabilityof lactic acid bacteria under acidic conditions.

[0035] Examples of products prepared according to the presentdouble-coating process will be specifically described below.

EXAMPLE 1

[0036] For Lactobacillus acidophilus CBT-LH powders which aredouble-coated, the protein aqueous solution was prepared by suspending 4kg of skim milk and 2 kg of isolated soybean proteins in 100 kg waterand added was an enzyme solution, which was prepared by dissolving 1.02g of proteolytic enzyme(Protease-N available from Amano Corp.) in 100 mlwater, to the protein solution at 55° C. and primary pH value 7.0.Hydrolysis reaction was allowed until the pH value decreases to 6.0within an enzyme-treating equipment with a low speed stirrer, athermostat and a pH control device.

[0037] After hydrolysis, added and dissolved were 5 kg glucose, 1 kgconcentrated meat extract, 0.5 kg yeast extract, 50 g dipotassiumphosphate, 50 g ammonium citrate, 50 g sodium acetate, 10 g magnesiumsulfate and 10 g manganese sulfate to the solution. This preparedsolution was fed into a 200 L anaerobic fermenter and sterilized at 121°C. for 15 minutes.

[0038] Afterward, the 2 L starter of Lactobacillus acidophilus CBT-LHwas cultivated for 12 hours while maintaining a pH level of 6.0 withammonia. Then, the bacteria cultures and residual protein wereprecipitated and concentrated after the solution was fed into acontinuous centrifuge at a flow rate of 60 L/Hr.

[0039] Separately, 10 L of cryoprotectant aqueous solution containing 1kg trehalose, 1 kg mannitol and 1 kg malto-dextrin was prepared andsterilized under pressure. In addition, 3 L polysaccharide aqueoussolution was prepared by dissolving 10 g xantan gum, 10 g cellulose and10 g levan, followed by sterilizing of the solution under pressure.

[0040] The concentrated bacteria cultures were introduced into a mixtureof the cryoprotectant and the polysaccharide solution prepared asdescribed above, then fed into a stirrer to form a homogenized solutionstirring at 5,000 RPM. This homogenized solution was frozen rapidly to−55° C. and lyophilized at a temperature ranging of 0 to 40° C.

[0041] During the centrifugal precipitation process, it was observedthat the water-semisoluble residual protein components enclosed thebacteria cells while the water-soluble peptide components were depositedon the cellular walls of the bacteria cells thereby coating the cultureswith protein. On the other hand, the polysaccharide components allowedthe bacteria cultures to form the bacteria culture group having verydense structures.

[0042] It was found that the double-coated lactic acid bacteria producedusing protein and the composition of xantan gum, cellulose and levanshowed improved stability in the acceleration test, acid-resistance andbile-resistance as compared to the non-coated lactic acid bacteria.Results of the above test are shown in Table 1. TABLE 1 Results foracid-resistance, bile-resistance and acceleration tests for thedouble-coated Lactobacillus acidophilus CBT-LH Acid-resistance testAcceleration test (in artificial gastric Bile-resistance test (in (40°C., 70% juice at pH 2.1) 0.5% Oxgall solution) relative humidity)Exposure Test Exposure Test Time Test time material Control timematerial Control elapsed material Control (min) (cfu/g) (cfu/g) (min)(cfu/g) (cfu/g) (day) (cfu/g) (cfu/g) 0 3.4 × 10¹¹ 3.3 × 10¹¹ 0 3.5 ×10¹¹ 3.4 × 10¹¹ 0 3.1 × 10¹¹ 3.2 × 10¹¹ 30 3.2 × 10¹¹ 2.3 × 10¹¹ 30 3.3× 10¹¹ 2.1 × 10¹¹ 10 2.9 × 10¹¹ 1.1 × 10¹¹ 60 3.1 × 10¹¹ 1.5 × 10⁹  603.2 × 10¹¹ 1.4 × 10¹¹ 20 2.6 × 10¹¹ 8.9 × 10¹⁰ 90 3.1 × 10¹¹ 1.2 × 10¹¹90 3.0 × 10¹¹ 1.1 × 10¹¹ 30 2.5 × 10¹¹ 1.0 × 10¹¹ 120 2.9 × 10¹¹ 9.3 ×10¹⁰ 120 2.9 × 10¹¹ 9.9 × 10¹⁰ 40 2.0 × 10¹¹ 9.8 × 10⁹  Survival 85.328.2 Survival 82.9 29.1 Survival 64.5 3.1 rate rate rate after afterafter 120 mins 120 mins 40 (%) (%) days (%)

EXAMPLE 2

[0043] For Streptococcus thermophilus CBT-ST powders which aredouble-coated, the protein aqueous solution was prepared by suspending 6kg of skim milk in 100 kg water and to it was added an enzyme solution,which was prepared by dissolving 6 g of proteolytic enzyme(Protease-Navailable from Amano Corp.) in 100 ml water, to the protein solution at55° C. and primary pH 7.0. Hydrolysis reaction was allowed until the pHvalue decreased to 6.2 within an enzyme-treating equipment with a lowspeed stirrer, a thermostat and a pH control device.

[0044] After hydrolysis, were added and dissolved 4 kg glucose, 0.5 kgconcentrated extract concentrate, 0.5 kg yeast extract, 50 g dipotassiumphosphate, 50 g ammonium citrate, 50 g sodium acetate, 20 g magnesiumsulfate and 5 g manganese sulfate to the solution. This preparedsolution was fed into a 200 L anaerobic fermenter and sterilized at 121°C. for 15 minutes.

[0045] Afterward, the 2 L starter of Streptococcus thermophilus CBT-STwas cultivated for 12 hours while maintaining a pH level of 6.0 withammonia. Then, the bacteria cultures and residual protein wereprecipitated and concentrated after the fermented solution was fed intoa continuous centrifuge at a flow rate of 60 L/Hr.

[0046] Separately, 10 L of cryoprotectant aqueous solution containing0.5 kg trehalose, 0.5 kg mannitol and 0.5 kg malto-dextrin and 1.5 Kgskim milk was prepared and sterilized under pressure. In addition, 3 Lof polysaccharide aqueous solution was prepared by dissolving 15 gxantan gum and 15 g cellulose, followed by sterilization of the solutionunder pressure.

[0047] The concentrated bacteria cultures were introduced into a mixtureof the cryoprotectant solution and the polysaccharide solution preparedas described above, then fed into a stirrer to form a homogenizedsolution stirring at 5,000 RPM. This homogenized solution was frozenrapidly to −55° C. and lyophilized at a temperature ranging of 0 to 40°C.

[0048] It was found that the double-coated lactic acid bacteria,produced by protein-coating using skim milk and polysaccharide-coatingusing xantan gum and cellulose, showed improved stability in theacceleration test, acid-resistance and bile-resistance as compared tothe non-coated lactic acid bacteria. Results of the above test are shownin Table 2. TABLE 2 Results for acid-resistance, bile-resistance andacceleration tests for double-coated Streptococcus thermophilus CBT-STAcid-resistance test Acceleration test (in artificial gastricBile-resistance test (40° C., 70% juice at pH 2.1) (in 0.5% Oxgallsolution) relative humidity) Exposure Test Exposure Test Time Test timematerial Control time material Control elapsed material Control (min)(cfu/g) (cfu/g) (min) (cfu/g) (cfu/g) (day) (cfu/g) (cfu/g) 0 2.0 × 10¹¹2.2 × 10¹¹ 0 2.7 × 10¹¹ 2.9 × 10¹¹ 0 2.6 × 10¹¹ 2.7 × 10¹¹ 30 1.9 × 10¹¹1.7 × 10¹¹ 30 2.7 × 10¹¹ 2.3 × 10¹¹ 10 2.5 × 10¹¹ 1.3 × 10¹¹ 60 1.8 ×10¹¹ 1.2 × 10¹¹ 60 2.5 × 10¹¹ 1.9 × 10¹¹ 20 2.5 × 10¹¹ 9.7 × 10¹⁰ 90 1.8× 10¹¹ 9.5 × 10¹⁰ 90 2.4 × 10¹¹ 1.5 × 10¹⁰ 30 2.4 × 10¹¹ 8.6 × 10¹⁰ 1201.8 × 10¹¹ 8.4 × 10¹⁰ 120 2.4 × 10¹¹ 9.2 × 10¹⁰ 40 2.2 × 10¹¹ 5.1 × 10¹⁰Survival 90.0 38.2 Survival 88.9 31.7 Survival 84.6 18.9 rate rate rateafter after after 120 mins 120 mins 40 (%) (%) days (%)

EXAMPLE 3

[0049] For Bifidobacterium longum CBT-BG powders which aredouble-coated, the protein aqueous solution was prepared by suspending 2kg of isolated soybean protein in 100 kg water and added was an enzymesolution, which was prepared by dissolving 5.4 g of proteolyticenzyme(Protease-N available from Amano Corp.) in 100 ml water, to theprotein solution at 55° C. and primary pH value 7.0. Hydrolysis reactionwas allowed until the pH value decreased to 6.2 within anenzyme-treating equipment with a low speed stirrer, a thermostat and apH control device.

[0050] After hydrolysis, added and dissolved were 4 kg glucose, 0.5 kgmeat extract concentrate, 0.5 kg yeast extract, 1 Kg casein peptone, 50g dipotassium phosphate, 50 g potassium phosphate, 10 g magnesiumsulfate, 1 g sodium chloride, and 10 g manganese sulfate into thesolution. This prepared solution was fed into a 200 L anaerobicfermenter and sterilized at 121° C. for 15 minutes.

[0051] Afterward, the 2 L starter of Bifidobacterium longum CBT-BG wascultivated for 15 hours while maintaining a pH level of 6.0 withammonia. The bacteria cultures and residual protein were precipitatedand concentrated after the fermented solution was fed into a continuouscentrifuge at a flow rate of 60 L/Hr.

[0052] The concentrated bacteria culture was introduced into the 10 L ofcryoprotectant aqueous solution, containing 0.5 kg trehalose, 0.5 kgmannitol and 0.5 kg malto-dextrin and 1.5 Kg skim milk, and mixedhomogeneously using a stirrer at 5,000 RPM. This homogenized solutionwas frozen rapidly to −55° C. and lyophilized at a temperature rangingof 0 to 40° C.

[0053] 10 g of frozen dried bacteria culture powder was introduced intoa polysaccharide solution prepared by dissolving 15 g xantan gum and 15g cellulose into 3 L of water and mixed homogeneously using stirrer at5,000 RPM, followed by freezing to −55° C. and lyophilizing under thecondition ranging of 0 to 40° C.

[0054] It was found that the double-coated lactic acid bacteria,produced by protein-coating using isolated soybean protein andpolysaccharide-coating using a combination of xantan gum and cellulose,showed improved stability in the acceleration test, acid-resistance andbile-resistance as compared to the non-coated lactic acid bacteria.Results of the above test are shown in Table 3. TABLE 3 Results foracid-resistance, bile-resistance and acceleration tests fordouble-coated Bifidobacterium longum CBT-BG Acid-resistance testAcceleration test (in artificial gastric Bile-resistance test (40° C.,70% juice at pH 2.1) (in 0.5% Oxgall solution) relative humidity)Exposure Test Exposure Test time Test time material Control timematerial Control elapsed material Control (min) (cfu/g) (cfu/g) (min)(cfu/g) (cfu/g) (day) (cfu/g) (cfu/g) 0 4.0 × 10¹¹ 3.8 × 10¹¹ 0 3.7 ×10¹¹ 3.6 × 10¹¹ 0 3.6 × 10¹¹ 4.0 × 10¹¹ 30 3.9 × 10¹¹ 2.3 × 10¹¹ 30 3.5× 10¹¹ 2.6 × 10¹¹ 10 3.3 × 10¹¹ 1.1 × 10¹¹ 60 3.8 × 10¹¹ 2.1 × 10¹¹ 603.4 × 10¹¹ 2.3 × 10¹¹ 20 2.9 × 10¹¹ 9.3 × 10¹⁰ 90 3.8 × 10¹¹ 1.3 × 10¹⁰90 3.2 × 10¹¹ 1.9 × 10¹⁰ 30 2.7 × 10¹¹ 3.7 × 10¹⁰ 120 3.5 × 10¹¹ 9.9 ×10¹⁰ 120 3.0 × 10¹¹ 9.8 × 10¹⁰ 40 2.3 × 10¹¹ 1.4 × 10¹⁰ Survival 87.526.1 Survival 81.3 27.2 Survival 63.9 3.5 rate rate rate after afterafter 120 mins 120 mins 40 (%) (%) days (%)

[0055] As illustrated above, it is shown that the double-coated lacticacid bacteria powders prepared by the method according to the presentinvention have advantages such as excellent storage stability,acid-resistance, bile-resistance and heat-resistance, as compared tonon-coated or primary-coated lactic acid bacteria.

[0056] The present invention is applicable in combination with manykinds of cryoprotectants and stabilizers depending on the properties ofthe used bacteria and can provide double-coated lactic acid bacteriathrough homogenization in an aqueous solution state and lyophilizationprocess thereby requiring no additional equipment and apparatus in theprocess for preparing the above double-coated lactic acid bacteria.

[0057] The present invention is also applicable to both concentratedbacterial cultures before drying and dried cultures thereby achievingimproved flexibility and compatibility of the production process. Inparticular, the present invention has enhanced the recovery rate of thelactic acid bacteria powders because of a superior survival rate of thebacteria cultures ranging of 50-90%, which improves productivity.

[0058] The lactic acid bacteria powders produced by the processaccording to the present invention show noticeably improvedheat-resistance, acid-resistance, bile-resistance and storage stability,as well as excellent viability within the intestines to exhibit superiorphysiological functions inherent to lactic acid bacteria, thereby havingmaximum efficiency in using the same.

What is claimed is:
 1. A process for preparing a double-coated lacticacid bacteria culture powders comprising the steps of: preparing aprotein aqueous solution; adding a proteolytic enzyme solution to theprotein aqueous solution for proteolysis; cultivating a lactic acidbacteria in the protein aqueous solution; concentrating the lactic acidbacteria culture using a centrifuge wherein the lactic acid bacteriaculture is coated with the protein; introducing the lactic acid bacteriaculture into a polysaccharide aqueous solution and mixing to form ahomogenized solution; and lyophilizing the homogenized solution.
 2. Theprocess set forth in claim 1, wherein the polysaccharide is selectedfrom the group consisting of Xantan gum, Cellulose, Levan and mixturesthereof.
 3. The process set forth in claim 1, wherein the proteinaqueous solution comprises one selected from the group consisting ofskim milk, isolated soybean protein and mixture thereof.
 4. The processset forth in claim 1, further comprising the step of mixing a glucose,an yeast extract, a concentrated meat extract and an ionic componentinto the protein aqueous solution before cultivating step.
 5. Theprocess set forth in claim 4, wherein the ionic component is selectedfrom the group consisting of ammonium citrate, sodium acetate,dipotassium phosphate, magnesium sulfate, manganese sulfate, sodiumchloride and mixtures thereof.
 6. The process set forth in claim 1,wherein the polysaccharide aqueous solution further comprises acryoprotectant.
 7. The process set forth in claim 6, wherein thecryoprotectant includes one selected from a group consisting oftrehalose, malto-dextrin, mannitol, skim milk and mixtures thereof.
 8. Aprocess for preparing a double-coated lactic acid bacteria culturepowders comprising the steps of: preparing a protein aqueous solution;adding a proteolytic enzyme to the protein aqueous solution forproteolysis; cultivating a lactic acid bacteria in the protein aqueoussolution; concentrating the lactic acid bacteria culture using acentrifuge wherein the lactic acid bacteria culture is coated with theprotein; mixing the lactic acid bacteria culture into a cryoprotectantaqueous solution homogeneously followed by lyophilization; introducingthe lyophilized lactic acid bacteria culture into a polysaccharideaqueous solution and mixing to form a homogenized solution; andlyophilizing the homogenized solution.
 9. A double-coated lactic acidbacteria produced by the process described in any one of claims 1-8.